CN110161149B - Method for detecting impurity a-tropine in tropisetron hydrochloride injection - Google Patents
Method for detecting impurity a-tropine in tropisetron hydrochloride injection Download PDFInfo
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- 238000002347 injection Methods 0.000 title claims abstract description 66
- 239000007924 injection Substances 0.000 title claims abstract description 66
- 229960003674 tropisetron hydrochloride Drugs 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000012535 impurity Substances 0.000 title claims abstract description 24
- XIEGSJAEZIGKSA-LUNMCBQDSA-N tropisetron hydrochloride Chemical compound Cl.C1=CC=C2C(C(=O)O[C@H]3C[C@H]4CC[C@@H](C3)N4C)=CNC2=C1 XIEGSJAEZIGKSA-LUNMCBQDSA-N 0.000 title abstract 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 66
- 238000001514 detection method Methods 0.000 claims abstract description 46
- 238000010812 external standard method Methods 0.000 claims abstract description 11
- 238000004817 gas chromatography Methods 0.000 claims abstract description 11
- 238000012360 testing method Methods 0.000 claims description 62
- ZNRGQMMCGHDTEI-ITGUQSILSA-N tropisetron Chemical compound C1=CC=C2C(C(=O)O[C@H]3C[C@H]4CC[C@@H](C3)N4C)=CNC2=C1 ZNRGQMMCGHDTEI-ITGUQSILSA-N 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 55
- 239000012085 test solution Substances 0.000 claims description 43
- 239000000523 sample Substances 0.000 claims description 32
- 239000012488 sample solution Substances 0.000 claims description 32
- 239000012088 reference solution Substances 0.000 claims description 13
- 238000007865 diluting Methods 0.000 claims description 12
- 238000001704 evaporation Methods 0.000 claims description 11
- 230000014759 maintenance of location Effects 0.000 claims description 10
- 238000005070 sampling Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000005303 weighing Methods 0.000 claims description 10
- 239000012159 carrier gas Substances 0.000 claims description 9
- 150000003254 radicals Chemical class 0.000 claims description 9
- 229960003688 tropisetron Drugs 0.000 claims description 9
- UIVFDCIXTSJXBB-ITGUQSILSA-N tropisetron Chemical compound C1=CC=C[C]2C(C(=O)O[C@H]3C[C@H]4CC[C@@H](C3)N4C)=CN=C21 UIVFDCIXTSJXBB-ITGUQSILSA-N 0.000 claims description 9
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000000463 material Substances 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 6
- 238000004809 thin layer chromatography Methods 0.000 abstract description 3
- 239000012141 concentrate Substances 0.000 abstract 1
- 230000004043 responsiveness Effects 0.000 abstract 1
- 238000010200 validation analysis Methods 0.000 description 23
- 239000013558 reference substance Substances 0.000 description 14
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 8
- 239000002202 Polyethylene glycol Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 238000013112 stability test Methods 0.000 description 6
- 239000011550 stock solution Substances 0.000 description 5
- 239000002994 raw material Substances 0.000 description 4
- 238000012418 validation experiment Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 239000012490 blank solution Substances 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 2
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- 230000015556 catabolic process Effects 0.000 description 2
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- 238000011084 recovery Methods 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000031649 Postoperative Nausea and Vomiting Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 239000005557 antagonist Substances 0.000 description 1
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- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
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- 230000003389 potentiating effect Effects 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/12—Preparation by evaporation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to a method for detecting impurity a-tropine alcohol in tropisetron hydrochloride injection, which develops a proper temperature-rising program and a proper chromatographic condition, so that alpha-tropine alcohol can be well separated from tropisetron hydrochloride, auxiliary materials and related impurities, and the detection of the alpha-tropine alcohol is not interfered by an auxiliary material peak and other impurity peaks in the injection, thereby having strong specificity; the invention firstly concentrates the injection, then dilutes the injection by methanol, and adopts a direct injection method to inject sample, thereby increasing the responsiveness of alpha-tropine in a gas chromatograph and improving the detection sensitivity, and the invention can detect the alpha-tropine residue of 0.09 mu g/mL, and provides guarantee for detecting trace alpha-tropine residue in the current detection sample; the method adopts the gas chromatography and calculates the content of the alpha-tropine according to an external standard method, greatly improves the quantitative capability of impurity detection and increases the controllability of the medicine quality compared with the common thin-layer chromatography in the prior art.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to a method for detecting impurity a-tropine in tropisetron hydrochloride injection.
Background
The tropisetron hydrochloride injection is one kind of peripheral nerve source and central nervous system 5-hydroxytryptamine 3 (5-HT) 3 ) Potent, highly selective competitive antagonists of receptors, primarily by selectively blocking presynaptic 5-HT of peripheral neurons 3 Receptor inhibition of the emetic reflex, and antiemetic action may be combined with its central 5-HT 3 Direct receptor blockade is associated with inhibition of the stimulation of the vagus nerve in the posterior region.
The original developer of tropisetron hydrochloride injection was Sandoz, switzerland, first marketed in the netherlands in 1992 under the trade name: navoban is used for preventing and treating nausea and vomiting caused by tumor radiotherapy and chemotherapy, and postoperative nausea and vomiting. In 1999 the product is first marketed in China with the trade name: european Booth.
The alpha-tropine is an important fragment for synthesizing the tropisetron hydrochloride raw material medicine and is also a degradation impurity of the tropisetron hydrochloride injection, and the degradation process is as follows:
according to the analysis, the alpha-tropine in the tropisetron hydrochloride injection has two source ways, and can be brought in from a tropisetron hydrochloride raw material medicine or generated by the degradation of the injection. Therefore, the detection of the impurity alpha-tropine in the tropisetron hydrochloride injection is particularly important.
However, alpha-tropine itself has no ultraviolet absorption, and cannot be analyzed and detected by a high performance liquid chromatography ultraviolet detector, and meanwhile, the concentration of inorganic salt as an auxiliary material in the tropisetron hydrochloride injection is high, and the analysis and detection cannot be performed by an evaporative light scattering detector. The method has the advantages that the thin-layer chromatography is adopted in the Chinese pharmacopoeia 2015 edition and the European pharmacopoeia 9.0 to detect the impurity alpha-tropine in the tropisetron hydrochloride raw material medicine, the method in the pharmacopoeia has the defects of poor sensitivity, semi-quantitative detection and poor repeatability, and more importantly, the method is high in concentration (40mg/ml) of a test sample, is only suitable for detection of the raw material medicine and cannot detect injection with determined concentration (the concentration of the tropisetron hydrochloride injection is 1 mg/ml).
However, in the prior art, other methods for detecting alpha-tropine in tropisetron hydrochloride injection generally have the problems of poor sensitivity, semi-quantitative detection and poor reproducibility, particularly have very obvious embodiment in the aspect of sensitivity, and the existing detection method has very high detection limit on the alpha-tropine in the tropisetron hydrochloride injection.
Therefore, the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection at present needs to be further improved.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting impurity a-tropine alcohol in tropisetron hydrochloride injection with high sensitivity and low detection limit aiming at the current situation of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for detecting impurity a-tropine in tropisetron hydrochloride injection adopts gas chromatography for detection, and is characterized in that: the method comprises the following steps of (1) preparing a test solution: taking the tropisetron hydrochloride injection, placing the tropisetron hydrochloride injection on a water bath, evaporating to dryness, dissolving with methanol, transferring to a measuring flask, and fixing the volume to prepare a solution containing 1-10 mg/mL of tropisetron;
(2) preparing a reference substance solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving and diluting to prepare a solution of 5-50 mu g/mL;
(3) and (3) detection: directly sampling the sample solution and the reference solution, and collecting chromatogram;
chromatographic conditions are as follows:
a chromatographic column: DB-WAX, 30 m.times.0.32 mm.times.0.5 μm;
column temperature: heating to 60-100 ℃ by a program and keeping the temperature for 3 minutes, then heating to 200 ℃ at the temperature of 10-20 ℃ per minute and keeping the temperature for 5 minutes;
carrier gas: n is a radical of 2 ；
The split ratio is as follows: 1: 1-3: 1;
flow rate: 1.0 ml/min-3.0 ml/min;
sample inlet temperature: 200-240 ℃;
a detector: hydrogen flame ionization detector, temperature: 240-260 ℃;
and (3) sample introduction mode: directly feeding a sample;
sample injection amount: 1 mul;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
(4) and (4) calculating a result:
content ═ C R /C x )×(A x /A R )，
In the formula: c R Concentration of control solution, C x Concentration of the test solution, A R Is the peak area of the alpha-tropine control, A x Is the peak area of alpha-tropine in the test sample.
Preferably, the concentration of the test solution is 1 mg/mL.
Preferably, the concentration of the control solution is 5 μ g/mL.
Preferably, in step (3), the column temperature is programmed to 80 ℃ for 3 minutes and then 15 ℃ per minute to 200 ℃ for 5 minutes.
Preferably, in step (3), the split ratio is 3: 1.
Preferably, in step (3), the flow rate is 1.5 mL/min.
Preferably, in step (3), the injection port temperature is 200 ℃.
Preferably, in step (3), the detector temperature is 250 ℃.
Compared with the prior art, the invention has the advantages that: the invention develops a proper temperature-rising program and chromatographic conditions, so that the alpha-tropine can be well separated from the tropisetron hydrochloride, the auxiliary materials and the related impurities, and the detection of the alpha-tropine is not interfered by the auxiliary material peak and other impurity peaks in the injection, thereby having strong specificity; aiming at the defect that the existing aqueous solution can not be directly injected, the injection is concentrated firstly and then diluted by methanol, so that the sample after treatment can be injected by adopting a direct injection method, the responsivity of the alpha-tropine alcohol in a gas chromatograph is increased, the detection sensitivity is improved, the invention can detect the residual alpha-tropine alcohol of 0.09 mu g/mL, which is equivalent to 0.009% of the concentration (1mg/mL) of the tropisetron hydrochloride injection, and provides guarantee for detecting the residual trace alpha-tropine alcohol in the existing detection sample; the method adopts the gas chromatography and calculates the content of the alpha-tropine according to an external standard method, greatly improves the quantitative capability of impurity detection and increases the controllability of the medicine quality compared with the common thin-layer chromatography in the prior art.
Drawings
FIG. 1 is a chromatogram of a control solution in example 1 of the present invention;
FIG. 2 is a chromatogram of a test solution in example 1 of the present invention;
FIG. 3 is a chromatogram of a proprietary test blank solution (methanol) in a validation test according to the present invention;
FIG. 4 is a chromatogram of a proprietary test blank adjuvant solution in a validation test of the present invention;
FIG. 5 is a chromatogram of a tropism test tropisetron hydrochloride solution in a validation test of the present invention;
FIG. 6 is a chromatogram of a proprietary test alpha-tropine alcohol solution in a validation test of the present invention;
FIG. 7 is a chromatogram of a limit of detection and limit of quantitation test limit solution in a validation test of the present invention;
FIG. 8 is a chromatogram of a detection limit solution of a detection limit and quantitation limit test in a validation test of the present invention;
FIG. 9 is a chromatogram of a control solution (0.3 μ g/ml) of the linearity and range tests in a validation experiment of the present invention;
FIG. 10 is a chromatogram of a control solution (2.0. mu.g/ml) of a linearity and range test in a validation test of the present invention;
FIG. 11 is a chromatogram of a control solution (4.0. mu.g/ml) of a linear and range assay in a validation assay of the present invention;
FIG. 12 is a chromatogram of a control solution (5.0 μ g/ml) of the linearity and range tests in a validation experiment of the present invention;
FIG. 13 is a chromatogram of a control solution (7.5 μ g/ml) of the linearity and range tests in a validation experiment of the present invention;
FIG. 14 is a chromatogram of a control solution (10.0 μ g/ml) of the linearity and range tests in a validation experiment of the present invention;
FIG. 15 is a standard curve for linear and range tests for alpha-tropine in a validation test according to the present invention;
FIG. 16 is a chromatogram of a sample solution applied during a precision test in a proof test according to the present invention;
FIG. 17 is a chromatogram of a sample solution applied during precision testing in a proof test according to the present invention;
FIG. 18 is a chromatogram of a sample solution loaded during a precision test in a proof test according to the present invention;
FIG. 19 is a chromatogram of a sample solution applied during precision testing in a proof test according to the present invention;
FIG. 20 is a chromatogram of a sample solution loaded during a precision test in a proof test according to the present invention;
FIG. 21 is a chromatogram of a sample solution loaded during a precision test in a proof test according to the present invention;
FIG. 22 is a chromatogram of a sample solution (low concentration) from an accuracy test in a validation test according to the present invention;
FIG. 23 is a chromatogram of a sample (low concentration) test solution applied in an accuracy test in a validation test according to the present invention;
FIG. 24 is a chromatogram of a sample solution loaded (at low concentration) in an accuracy test in a validation test according to the present invention;
FIG. 25 is a chromatogram of a sample solution loaded (of intermediate concentration) from an accuracy test in a validation test according to the present invention;
FIG. 26 is a chromatogram of a sample solution from an accuracy test of the present invention;
FIG. 27 is a chromatogram of a sample solution loaded (of medium concentration) in an accuracy test in a validation test according to the present invention;
FIG. 28 is a chromatogram of a sample solution (high concentration) from an accuracy test in a validation test according to the present invention;
FIG. 29 is a chromatogram of a sample solution (high concentration) from an accuracy test in a validation test according to the present invention;
FIG. 30 is a chromatogram of a sample solution (high concentration) from an accuracy test in a validation test according to the present invention;
FIG. 31 is a chromatogram of a sample solution (0 hour) loaded from a solution stability test in a validation test according to the present invention;
FIG. 32 is a chromatogram of a sample solution (2 hours) loaded during a solution stability test in a validation test according to the present invention;
FIG. 33 is a chromatogram of a sample solution (4 hours) loaded from a solution stability test in a validation test of the present invention;
FIG. 34 is a chromatogram of a sample solution (6 hours) loaded from a solution stability test in a validation test of the present invention;
FIG. 35 is a chromatogram of a sample solution (8 hours) loaded during a solution stability test in a validation test according to the present invention;
FIG. 36 is a chromatogram of a sample solution (24 hours) loaded in a solution stability test in a validation test of the present invention.
Detailed Description
The invention is described in further detail below with reference to the following examples of the drawings.
Example 1:
the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection adopts gas chromatography for detection, and comprises the following steps:
preparing a test solution: taking the tropisetron hydrochloride injection, placing the tropisetron hydrochloride injection on a water bath, evaporating to dryness, dissolving with methanol, transferring to a measuring flask, and fixing the volume to prepare a solution containing 1mg/mL of tropisetron;
preparation of a reference solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving, and diluting to prepare a solution of 5 mu g/mL;
directly sampling a sample solution and a reference solution respectively, and collecting a chromatogram;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
chromatographic conditions are as follows:
a chromatographic column: polyethylene glycol is stationary liquid or capillary column with similar polarity, DB-WAX is selected in the embodiment, and the diameter is 30m multiplied by 0.32mm multiplied by 0.5 mu m;
column temperature: keeping the temperature at 80 ℃ for 3 minutes, then raising the temperature to 200 ℃ at 15 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of 2 ；
The split ratio is as follows: 3: 1;
flow rate: 1.5 mL/min;
sample inlet temperature: 200 ℃;
detector (FID) temperature: 250 ℃;
sample injection amount: 1 μ L.
FIG. 1 is a chromatogram of a control solution, wherein 12.070min is the alpha-tropine peak; FIG. 2 is a chromatogram of a sample solution. Determining the content of the alpha-tropine according to the peak area of the alpha-tropine in the test solution, wherein the detection result is undetected.
Example 2:
the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection adopts gas chromatography for detection, and comprises the following steps:
preparing a test solution: evaporating the tropisetron hydrochloride injection on a water bath to dryness, dissolving the tropisetron hydrochloride injection with methanol, transferring the dissolved tropisetron hydrochloride injection into a measuring flask, and fixing the volume to prepare a solution containing 1mg/mL of tropisetron;
preparing a reference substance solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving and diluting to prepare a solution of 5 mu g/mL;
directly sampling a sample solution and a reference solution respectively, and collecting a chromatogram;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
chromatographic conditions are as follows:
and (3) chromatographic column: polyethylene glycol is stationary liquid or capillary column with similar polarity, and DB-WAX is recommended, 30m × 0.32mm × 0.5 μm;
column temperature: keeping the temperature at 100 ℃ for 3 minutes, then raising the temperature to 200 ℃ at 10 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of 2 ；
The split ratio is as follows: 3: 1;
flow rate: 1.5 mL/min;
sample inlet temperature: 200 ℃;
detector (FID) temperature: 250 ℃;
sample injection amount: 1 μ L.
Determining the content of the alpha-tropine according to the peak area of the alpha-tropine in the test solution, wherein the detection result is undetected.
Example 3:
the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection adopts gas chromatography for detection, and comprises the following steps:
preparing a test solution: evaporating the tropisetron hydrochloride injection on a water bath to dryness, dissolving the tropisetron hydrochloride injection with methanol, transferring the dissolved tropisetron hydrochloride injection into a measuring flask, and fixing the volume to prepare a solution containing 1mg/mL of tropisetron;
preparation of a reference solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving, and diluting to prepare a solution of 5 mu g/mL;
directly sampling the sample solution and the reference solution, and collecting chromatogram;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
chromatographic conditions are as follows:
and (3) chromatographic column: polyethylene glycol is stationary liquid or capillary column with similar polarity, and DB-WAX is recommended, 30m × 0.32mm × 0.5 μm;
column temperature: keeping the temperature at 60 ℃ for 3 minutes, then heating to 200 ℃ at 20 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of hydrogen 2 ；
The split ratio is as follows: 3: 1;
flow rate: 1.5 mL/min;
sample inlet temperature: 200 ℃;
detector (FID) temperature: 250 ℃;
sample injection amount: 1 μ L.
Determining the content of the alpha-tropine according to the peak area of the alpha-tropine in the test solution, wherein the detection result is undetected.
Example 4:
the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection adopts gas chromatography for detection, and comprises the following steps:
preparing a test solution: evaporating the tropisetron hydrochloride injection on a water bath to dryness, dissolving the tropisetron hydrochloride injection with methanol, transferring the dissolved tropisetron hydrochloride injection into a measuring flask, and fixing the volume to prepare a solution containing 1mg/mL of tropisetron;
preparation of a reference solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving, and diluting to prepare a solution of 5 mu g/mL;
directly sampling a sample solution and a reference solution respectively, and collecting a chromatogram;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
chromatographic conditions are as follows:
a chromatographic column: polyethylene glycol is stationary liquid or capillary column with similar polarity, and preferably DB-WAX, 30m × 0.32mm × 0.5 μm;
column temperature: keeping the temperature at 80 ℃ for 3 minutes, then raising the temperature to 200 ℃ at 15 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of 2 ；
The split ratio is as follows: 1: 1;
flow rate: 1.0 mL/min;
sample inlet temperature: 200 ℃;
detector (FID) temperature: 250 ℃;
sample introduction amount: 1 μ L.
Determining the content of the alpha-tropine according to the peak area of the alpha-tropine in the test solution, wherein the detection result is undetected.
Example 5:
the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection adopts gas chromatography for detection, and comprises the following steps:
preparing a test solution: taking the tropisetron hydrochloride injection, placing the tropisetron hydrochloride injection on a water bath, evaporating to dryness, dissolving with methanol, transferring to a measuring flask, and fixing the volume to prepare a solution containing 1mg/mL of tropisetron;
preparing a reference substance solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving and diluting to prepare a solution of 5 mu g/mL;
directly sampling the sample solution and the reference solution, and collecting chromatogram;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
chromatographic conditions are as follows:
a chromatographic column: polyethylene glycol is stationary liquid or capillary column with similar polarity, and preferably DB-WAX, 30m × 0.32mm × 0.5 μm;
column temperature: keeping the temperature at 80 ℃ for 3 minutes, then heating to 200 ℃ at 15 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of 2 ；
The split ratio is as follows: 3: 1;
flow rate: 3.0 mL/min;
sample inlet temperature: 240 ℃;
detector (FID) temperature: 250 ℃;
sample introduction amount: 1 μ L.
Determining the content of the alpha-tropine according to the peak area of the alpha-tropine in the test solution, wherein the detection result is undetected.
Example 6:
the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection adopts gas chromatography for detection, and comprises the following steps:
preparing a test solution: evaporating the tropisetron hydrochloride injection on a water bath to dryness, dissolving the tropisetron hydrochloride injection with methanol, transferring the dissolved tropisetron hydrochloride injection into a measuring flask, and fixing the volume to prepare a solution containing 10mg/mL of tropisetron;
preparing a reference substance solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving and diluting to prepare a solution of 50 mu g/mL;
directly sampling the sample solution and the reference solution, and collecting chromatogram;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
chromatographic conditions are as follows:
and (3) chromatographic column: polyethylene glycol is stationary liquid or capillary column with similar polarity, and DB-WAX is recommended, 30m × 0.32mm × 0.5 μm;
column temperature: keeping the temperature at 80 ℃ for 3 minutes, then raising the temperature to 200 ℃ at 15 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of hydrogen 2 ；
The split ratio is as follows: 3: 1;
flow rate: 1.5 mL/min;
sample inlet temperature: 200 ℃;
detector (FID) temperature: 260 ℃;
sample introduction amount: 1 μ L.
Determining the content of the alpha-tropine according to the peak area of the alpha-tropine in the test solution, wherein the detection result is undetected.
Example 7:
the method for detecting the impurity a-tropine in the tropisetron hydrochloride injection adopts gas chromatography for detection, and comprises the following steps:
preparing a test solution: evaporating tropisetron hydrochloride injection on a water bath to dryness, dissolving with methanol, transferring to a measuring flask, adding alpha-tropine alcohol to prepare 0.3 mu g/mL alpha-tropine alcohol solution, fixing the volume, and shaking up;
preparing a reference substance solution: taking a proper amount of alpha-tropine reference substance, precisely weighing, adding methanol for dissolving and diluting to prepare a solution of 5 mu g/mL;
directly sampling a sample solution and a reference solution respectively, and collecting a chromatogram;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
chromatographic conditions are as follows:
a chromatographic column: polyethylene glycol is stationary liquid or capillary column with similar polarity, DB-WAX is selected in the embodiment, and the thickness is 30m multiplied by 0.32mm multiplied by 0.5 mu m;
column temperature: keeping the temperature at 80 ℃ for 3 minutes, then raising the temperature to 200 ℃ at 15 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of 2 ；
The split ratio is as follows: 3: 1;
flow rate: 1.5 mL/min;
sample inlet temperature: 200 ℃;
detector (FID) temperature: 250 ℃;
sample introduction amount: 1 μ L.
The content of the alpha-tropine is determined according to the peak area of the alpha-tropine in the test solution, and the detection result shows that the detection limit is 0.09 mu g/mL.
In order to confirm the accuracy and sensitivity of the detection method of the present invention, the detection method of the present invention was subjected to a validation test in methodology:
taking example 1 as an example, methodological verification was performed respectively in terms of specificity, detection and quantitation limits, linearity and range, precision, accuracy, and solution stability, and the results were as follows:
(1) specificity
Taking blank solution (methanol), blank auxiliary materials, tropisetron hydrochloride and alpha-tropine positioning solution, and injecting 1 needle of each sample. The results show (fig. 3-6) that the blank solution, the blank auxiliary material and the tropisetron hydrochloride have no interference at the alpha-tropine peak-out position.
Specificity results
(2) Detection limit and quantification limit
Taking a proper amount of alpha-tropine reference substance, precisely weighing, dissolving and diluting with a diluent to prepare a solution of 0.1mg/ml, and then diluting downwards step by step. The concentration is determined as the limit of quantitation when the signal-to-noise ratio is not less than 10, and as the limit detection concentration when the signal-to-noise ratio is not less than 3.
Quantitative limit, detection limit result
The results show (FIG. 7, FIG. 8), the limit of quantitation of the method is 0.31. mu.g/ml, the limit of detection is 0.09. mu.g/ml, and the method has high sensitivity.
(3) Linearity and range
Taking the alpha-tropine control stock solution, sequentially diluting the alpha-tropine control stock solution into solutions with the concentrations of 0.3 mu g/ml, 2.0 mu g/ml, 4.0 mu g/ml, 5.0 mu g/ml, 7.5 mu g/ml and 10.0 mu g/ml, and detecting according to a method to obtain the linear relation of the concentrations.
As can be seen from FIGS. 9 to 15, in the concentration range of α -tropine from the limit of quantitation (0.3 μ g/ml) to 10.0 μ g/ml (200% of the limit), the α -tropine peak area is linearly related to the concentration, and the linear correlation coefficient R is 2 And was 0.9993.
(4) Precision degree
Taking the tropisetron hydrochloride injection, placing the tropisetron hydrochloride injection in an evaporation dish, evaporating the tropisetron hydrochloride injection on a water bath to dryness, dissolving the tropisetron hydrochloride injection with a diluent, transferring the tropisetron hydrochloride injection into a measuring flask, adding the alpha-tropine stock solution to dilute the tropisetron hydrochloride injection to a scale, fixing the volume, and shaking the tropisetron hydrochloride injection evenly. And 6 parts of sample adding test solution are prepared repeatedly, and the result is measured according to the method.
Results of precision verification
The results show (figures 16-21) that the retention time of the alpha-tropine is consistent for 6 parts of the repetitive solution, and the RSD of the content of 6 parts of the test solution meets the requirement (the RSD is less than or equal to 10 percent), which proves that the method has good precision.
(5) Accuracy of
Taking the alpha-tropine stock solution, respectively adding the alpha-tropine stock solution into a test sample to prepare low, medium and high sample concentration solutions, and determining the result according to a method.
Accuracy verification result
From the test results (fig. 22 to 30), it is understood that the average recovery rate of α -tropine in the low, medium and high concentration test solutions is 104.51% to 110.84%, which meets the recovery rate requirement (80% to 120%), and the method is excellent in accuracy.
(6) Stability of solution
Taking the sample-adding test sample solution, standing for 24 hours, and respectively sampling and detecting at different time periods, wherein the results are as follows:
results of solution stability verification
The results show (FIGS. 31-36) that the control solution and the sample solution of alpha-tropine are stable within 24 hours.
Claims (1)
1. A method for detecting impurity a-tropine in tropisetron hydrochloride injection is characterized in that gas chromatography is adopted for detection, and the method comprises the following steps: comprises the following steps
(1) Preparing a test solution: placing the tropisetron hydrochloride injection on a water bath, evaporating to dryness, dissolving with methanol, transferring to a measuring flask, and fixing the volume to prepare a solution containing 1-10 mg/mL of tropisetron;
(2) preparation of a reference solution: taking a proper amount of alpha-tropine control, precisely weighing, adding methanol to dissolve and diluting to prepare a solution of 5-50 mug/mL;
(3) and (3) detection: directly sampling a sample solution and a reference solution respectively, and collecting a chromatogram;
chromatographic conditions are as follows:
and (3) chromatographic column: DB-WAX, 30m × 0.32mm × 0.5 μm;
column temperature: keeping the temperature at 80 ℃ for 3 minutes, then heating to 200 ℃ at 15 ℃ per minute, and keeping the temperature for 5 minutes;
carrier gas: n is a radical of 2 ；
The split ratio is as follows: 3: 1;
flow rate: 1.5 ml/min;
sample inlet temperature: 200 ℃;
a detector: hydrogen flame ionization detector, temperature: 250 ℃;
and (3) sample introduction mode: directly feeding a sample;
sample introduction amount: 1 mu l;
if a chromatographic peak with the same retention time as that of the alpha-tropine exists in a chromatogram of the test solution, determining the content of the alpha-tropine in the test solution by calculating the peak area according to an external standard method;
(4) and (4) calculating a result:
content = (C) R /C x ）×(A x /A R ），
In the formula: c R Concentration of control solution, C x As the concentration of the test solution, A R Peak area for alpha-tropine control, A x Is the peak area of alpha-tropine in the test sample;
the concentration of the test solution is 1 mg/mL;
the concentration of the control solution is 5 mug/mL.
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| CN102495170A (en) * | 2011-11-26 | 2012-06-13 | 山东齐都药业有限公司 | Method for detecting alpha-tropine in tropisetron hydrochloride injection |
| CN102565226A (en) * | 2011-12-30 | 2012-07-11 | 山东齐都药业有限公司 | Detection method for alpha-tropine as impurity in hydrochloric acid tropisetron |
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